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Fig. 4 | BMC Cancer

Fig. 4

From: TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway

Fig. 4

TNF-α promotes AP-1 binding to the VEGF-D promoter. a. The two predicted putative AP1-binding sites contained in the −444 to −325 nt region of VEGF-D promoter are underlined (AP-1(1) in the nucleotide region −401 to -393 nt; AP-1(2) in the −345 to -337 nt). b. The effect of mutation of the AP-1 binding sites on the activity of VEGF-D promoter. Both of the two mutated constructs, PGL4-AP-1mut1 and PGL4-AP-1mut2, exhibited lower activities than the non-mutated construct PGL4-444. Furthermore, the activity weakened when the two sites were mutated simultaneously. The trend persisted upon treatment with TNF-α (50 ng/ml) (mutants, indicated with the × mark, are depicted schematically on the left; *P < 0.05). c, d. EMSA of AP-1. The nuclear extracts from NOZ cells could bind the biotin-labeled probes (lane 2). The competition assay revealed that pre-incubation with the cold probes (lane 3) but not the cold mutated probes (lane 5) diminished the intensity of the bands. TNF-α enhanced the combined effect of the nuclear extracts and the two AP-1-binding sites (lane 4). e, f. ChIP assay. Chromatin from NOZ or GBC-SD cells was immunoprecipitated with the anti-AP-1 antibody. The total extracted DNA (Input) and the immunoprecipitated samples were PCR-amplified using primers specific to the regions of the VEGF-D promoter containing the AP-1(1) binding site (119 bp) and AP-1(2) binding site (150 bp). A normal rabbit IgG and no antibody sample were also included as controls. Another experiment group was treated with 50 ng⁄ mL of TNF-α (bottom row), and TNF-α enhanced the intensity of the input and anti-AP-1 bands

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